Ketamine’s acute effects on negative brain states are mediated through distinct altered states of consciousness in humans

Ketamine commonly and rapidly induces dissociative and other altered states of consciousness (ASCs) in humans. However, the neural mechanisms that contribute to these experiences remain unknown. We used functional neuroimaging to engage key regions of the brain’s affective circuits during acute ketamine-induced ASCs within a randomized, multi-modal, placebo-controlled design examining placebo, 0.05 mg/kg ketamine, and 0.5 mg/kg ketamine in nonclinical adult participants (NCT03475277). Licensed clinicians monitored infusions for safety. Linear mixed effects models, analysis of variance, t-tests, and mediation models were used for statistical analyses. Our design enabled us to test our pre-specified primary and secondary endpoints, which were met: effects of ketamine across dose conditions on (1) emotional task-evoked brain activity, and (2) sub-components of dissociation and other ASCs. With this design, we also could disentangle which ketamine-induced affective brain states are dependent upon specific aspects of ASCs. Differently valenced ketamine-induced ASCs mediated opposing effects on right anterior insula activity. Participants experiencing relatively higher depersonalization induced by 0.5 mg/kg of ketamine showed relief from negative brain states (reduced task-evoked right anterior insula activity, 0.39 SD). In contrast, participants experiencing dissociative amnesia showed an exacerbation of insula activity (0.32 SD). These results in nonclinical participants may shed light on the mechanisms by which specific dissociative states predict response to ketamine in depressed individuals.

numbers of males and females to control for the self-reported biological sex of participants.We included biological sex as a confounder for linear mixed models that were used to examine ketamine-induced dose-dependent effects.However, we do acknowledge with our current sample size we might be under-powered to examine sex differences in the acute impact of ketamine on human neural circuit function.

Addressing data missingness
For each subject's questionnaire data under a certain dose condition, if the missing items were fewer than 10% of the total item numbers of the questionnaire, we replaced missing items with the group mean of that dose condition.This brought the sample size of CADSS to be n = 13, n = 12, n = 13 for placebo, 0.05 mg/kg, and 0.5 mg/kg and of 5D-ASC to be n = 13 for all three drug visits.

Linear mixed effects and t-test analyses of dose-dependent effects of ketamine on dissociation and other ASCs
For Clinician-Administered Dissociative States Scale (CADSS) subscales assessed at T = 0 min and 40 mins, the linear mixed effects model (LMM) included dose (placebo, 0.05 mg/kg, or 0.5 mg/kg), time (T = 0 min and 40 mins), and the dose-by-time interaction as fixed effects and subject as the random effect.For 5-Dimensional Altered States of Consciousness (5D-ASC) subscales collected only post-drug administration, the LMM included only dose as the fixed effect and subject as the random effect.For post-hoc paired t-tests between drug dose conditions and for later mediation analysis, we derived the post-versus pre-infusion change of each CADSS subcomponent.

Anatomical Data Preprocessing
A total of 1 T1-weighted (T1w) image found within the input BIDS dataset.The T1-weighted (T1w) image was corrected for intensity non-uniformity (INU) with N4BiasFieldCorrection 8 , distributed with ANTs 2.3.3 9 (RRID:SCR_004757), and used as T1w-reference throughout the workflow.The T1w-reference was then skull-stripped with a Nipype implementation of the antsBrainExtraction.sh workflow (from ANTs), using OASIS30ANTs as target template.Brain tissue segmentation of cerebrospinal fluid (CSF), white-matter (WM) and gray-matter (GM) was performed on the brain-extracted T1w using fast (FSL 5.0.9,RRID:SCR_002823 10 ).Brain surfaces were reconstructed using recon-all (FreeSurfer 6.0.1, RRID:SCR_001847 11 ) and the brain mask estimated previously was refined with a custom variation of the method to reconcile ANTs-derived and FreeSurfer-derived segmentations of the cortical gray-matter of Mindboggle (RRID:SCR_002438

Functional Data Preprocessing
For each of the BOLD runs found per subject (across all tasks and sessions), the following preprocessing was performed.First, a reference volume and its skull-stripped version were generated by aligning and averaging 1 single-band references (SBRefs).Susceptibility distortion correction (SDC) was omitted.The BOLD reference was then co-registered to the T1w reference using bbregister (FreeSurfer) which implements boundary-based registration 15 .Co-registration was configured with six degrees of freedom.Head-motion parameters with respect to the BOLD reference (transformation matrices, and six corresponding rotation and translation parameters) are estimated before any spatiotemporal filtering using mcflirt (FSL 5.0.9 16 ).First, a reference volume and its skull-stripped version were generated using a custom methodology of fMRIPrep.
The BOLD time-series were resampled onto the following surfaces (FreeSurfer reconstruction nomenclature): fsnative, fsaverage.The BOLD time-series (including slice-timing correction when applied) were resampled onto their original, native space by applying the transforms to correct for head-motion.These resampled BOLD time-series will be referred to as preprocessed BOLD in original space, or just preprocessed BOLD.The BOLD time-series were resampled into several standard spaces, correspondingly generating the following spatially normalized, preprocessed BOLD runs: MNI152Nlin6Asym, MNI152Nlin2009cAsym. First, a reference volume and its skullstripped version were generated using a custom methodology of fMRIPrep.Grayordinates files 17 containing 91k samples were also generated using the highest resolution fsaverage as intermediate standardized surface space.Automatic removal of motion artifacts using independent component analysis (ICA-AROMA, 18 ) was performed on the preprocessed BOLD on MNI space time-series after removal of non-steady state volumes and spatial smoothing with an isotropic, Gaussian kernel of 6mm FWHM (full-width half-maximum).Corresponding "nonaggressively" denoised runs were produced after such smoothing.Additionally, the "aggressive" noise-regressors were collected and placed in the corresponding confounds file.Several confounding time-series were calculated based on the preprocessed BOLD: framewise displacement (FD), DVARS and three region-wise global signals.FD was computed using two formulations following Power (absolute sum of relative motions 19 ) and Jenkinson (relative root mean square displacement between affines 16 ).FD and DVARS are calculated for each functional run, both using their implementations in Nipype (following the definitions by 19 ).The three global signals are extracted within the CSF, the WM, and the whole-brain masks.Additionally, a set of physiological regressors were extracted to allow for component-based noise correction (CompCor 20 ).Principal components are estimated after high-pass filtering the preprocessed BOLD time-series (using a discrete cosine filter with 128s cut-off) for the two CompCor variants: temporal (tCompCor) and anatomical (aCompCor).tCompCor components are then calculated from the top 2% variable voxels within the brain mask.For aCompCor, three probabilistic masks (CSF, WM, and combined CSF+WM) are generated in anatomical space.The implementation differs from that of Behzadi et al. in that instead of eroding the masks by 2 pixels on BOLD space, the aCompCor masks are subtracted a mask of pixels that likely contain a volume fraction of GM.This mask is obtained by dilating a GM mask extracted from the FreeSurfer's aseg segmentation, and it ensures components are not extracted from voxels containing a minimal fraction of GM.
Finally, these masks are resampled into BOLD space and binarized by thresholding at 0.99 (as in the original implementation).Components are also calculated separately within the WM and CSF masks.For each CompCor decomposition, the k components with the largest singular values are retained, such that the retained components' time series are sufficient to explain 50 percent of variance across the nuisance mask (CSF, WM, combined, or temporal).The remaining components are dropped from consideration.The head-motion estimates calculated in the correction step were also placed within the corresponding confounds file.The confound time series derived from head motion estimates and global signals were expanded with the inclusion of temporal derivatives and quadratic terms for each 21 .Frames that exceeded a threshold of 0.5 mm FD or 1.5 standardized DVARS were annotated as motion outliers.All resampling's can be performed with a single interpolation step by composing all the pertinent transformations (i.e., head-motion transform matrices, susceptibility distortion correction when available, and coregistrations to anatomical and output spaces).Gridded (volumetric) resampling was performed using antsApplyTransforms (ANTs), configured with Lanczos interpolation to minimize the smoothing effects of other kernels 22 .Non-gridded (surface) resampling was performed using mri_vol2surf (FreeSurfer).
Many internal operations of fMRIPrep use Nilearn 0.6.2 23(RRID:SCR_001362), mostly within the functional processing workflow.For more details of the pipeline, see the section corresponding to workflows in fMRIPrep's documentation.

Copyright Waiver
The above boilerplate text was automatically generated by fMRIPrep with the express intention that users should copy and paste this text into their manuscripts unchanged.It is released under the CC0 license.The boilerplate material begins at the section titled: Image Preprocessing.

Quality control procedure
Quality control diagnostics included visual inspection of the raw fMRI timeseries for artifacts and signal dropout, and a review of the fMRIprep summary report for each participant.Participants' data were excluded if more than 25% (37/148) of time points were detected as motion spikes.
Volumes with frame-wise displacement >0.5 mm or std DVARS >1.5 are defined as motion spikes.One participant's data for the 0.05 mg/kg was excluded.One participant was not able to complete brain scans due to nausea under the 0.5 mg/kg condition.One participant was unreachable after the completion of the first two scan visits (placebo and 0.5 mg/kg) and is missing the 0.05 mg/kg data.This resulted in n = 13, 11, and 12 for placebo, 0.05mg/kg, and 0.5mg/kg conditions, respectively.

Extracting behavioral performance
Accuracy, measured by number of button presses with reaction time (RT) > 200ms, and the mean RT of correct presses, were extracted from each subject's behavior data recorded from a button box.

Facial Expression of Emotion Task (FEET) data analysis of dose-dependent effects of ketamine on behavioral performance
To examine dose-dependent effects of ketamine on individuals' behavioral performance during FEET, we used linear mixed effects models (LMMs) with accuracy or mean RT as the dependent variable and with dose (placebo, 0.05 mg/kg, or 0.5 mg/kg) as the fixed effect.

Definition of region of interest (ROI)
As established in our previous work 24 , we defined ROIs with an automated meta-analysis approach using neurosynth.org.Specifically, we used Neurosynth uniformity (previously called forward inference) maps with a false discovery rate (FDR) threshold of .01 for the search terms of anterior insula, amygdala, subgenual anterior cingulate cortex (sgACC), and dorsal anterior cingulate cortex (dACC).We imposed a restriction that the peak of the ROIs should have a minimum z-score of 6.For the anterior insula, we also excluded voxels with a z-score <5 to keep only the most relevant voxels spatially located in the anterior portion of the insula via visual inspection.For the amygdala, Neurosynth maps were restricted by anatomically defined boundaries from the Automated Anatomical Labeling atlas.

Sensitivity analysis of dose-dependent effects of ketamine on anterior insula and amygdala activity in response to threat confounded by behavioral performance
As additional sensitivity analyses, we added the accuracy (button press number) and mean RT as covariates of no interest when examining ketamine's dose-dependent effect on FEET activations in selected ROIs.

Sensitivity analysis of dose-dependent effects of ketamine on dissociation and altered states of consciousness, and on anterior insula and amygdala activity in response to threat after controlling for the order of drug visits
To test whether the order of drug visits had an impact on our current finding, we created a categorical variable representing the six drug visit orders: i) placebo, 0.05 mg/kg, 0.5 mg/kg; ii) placebo, 0.5 mg/kg, 0.05 mg/kg; iii) 0.05 mg/kg, placebo, 0.5 mg/kg; iv) 0.05 mg/kg, 0.5 mg/kg, placebo; v) 0.5 mg/kg, placebo, 0.05 mg/kg; and vi) 0.5 mg/kg, 0.05 mg/kg, placebo.As another set of sensitivity analyses, we added the categorical order variable to all linear mixed models used for testing ketamine induced dose-dependent effects.

Mediation analysis of heart rate and blood pressure on ketamine's effects on amygdala and insula activity in response to threat
We monitored vital signs, including heart rate and systolic and diastolic blood pressure throughout each study visit.As an exploratory analysis, we also examined whether they mediated ketamine's effects on amygdala and insula activity in response to threat.

Intra-subject correlation analysis of subcomponents of dissociation and other altered states of consciousness
To explore the direct association between dissociation and other altered states of consciousness, repeated-measures correlation analyses were run using the rmcorr package (https://cran.rproject.org/package=rmcorr). Unlike conventional inter-subject correlation analysis, the repeated measures correlation analysis calculates intra-subject correlations, which in our case, reflects how different subcomponents of dissociation and other altered states of consciousness co-vary across visits.As with the mediation analysis, we did not include the 0.05 mg/kg condition in the correlation analysis because no significant change was observed for this condition compared to placebo.FDR correction was implemented to control for false positives.

Intra-correlation of subcomponents of dissociation and other altered states of consciousness
Although the dissociation subcomponents depersonalization and amnesia were correlated with derealization (Repeated Measures Correlation; depersonalization to derealization r = 0.93, pFDR < 0.001; amnesia to derealization r = 0.66, pFDR = 0.05), they were not correlated with each other (Repeated Measures Correlation r = 0.52, pFDR = 0.13), indicating that they may map onto different factors of ketamine-related experiences.As expected, greater amnesia was associated with greater fear/anxiety, measured by the 5-Dimensional Altered States of Consciousness (5D-ASC) rating scale (Repeated Measures Correlation r = 0.75, pFDR = 0.02).The standardized beta coefficient values of the ACME reflect the effect size of the mediation, in our case indicating ketamine at 0.5 mg/kg dose induced change of neural activity in the magnitude of standard deviation (SD) through the mediating effect of dissociation and other ASCs.an MRI-safe machine.c.Multi-modal measures were collected prior to and after infusion.Permission was obtained from the individuals shown in the photographs for use of their images.Several sub-figures show the results of comparing outcomes between the three drug conditions with regard to d. dissociative depersonalization, e. dissociative derealization, f. altered states of bliss, g. dissociative amnesia, h.altered states of anxiety, and i. impaired control and cognition.Participants' quotes related to specific subcomponents of dissociation and altered states of consciousness are shown within these sub-figures.j.Right anterior insula activity in response to threat faces.k.Right amygdala activity in response to threat faces.One-way repeated Analysis of Variance was used to analyze the data.Adjustments for multiple comparisons were made.In each boxplot, the central thick black line represents the median, color shaded boxes represent the first and third quartiles (the 25 th and 75 th percentiles), and the whiskers extend no further than 1.5 times of the distance between the first and third quartiles.*: p <0.05, **: p < 0.01, ***: p<0.001.Source data are provided as a Source Data file.

Suppl. Figure 3. Behavioral performances during the nonconscious Facial Expression of Emotion Task (FEET)
Accuracy (number of correct presses) for n=13 nonclinical participants under Threat (a.), Neutral (b.), and Threat vs Neutral (c.).Average reaction time (RT) of the correct presses under Threat (d.), Neutral (e.), and Threat vs Neutral (f.).One-way repeated Analysis of Variance was used to analyze the data.In each boxplot, the central thick black line represents the median, color shaded boxes represent the first and third quartiles (the 25 th and 75 th percentiles), and the whiskers extend no further than 1.5 times of the distance between the first and third quartiles.Source data are provided as a Source Data file.

Suppl. Figure 1 .
Outcomes regarding altered states of consciousness (ASCs) hypothesized to associate with negative affective brain states.a. Randomized dose (X) (n=13 nonclinical participants).b.Licensed study clinicians monitored heart rate, blood pressure, and pulse oximetry during infusion and scan with

Figure 4 .
Experiences of anxiety mediate distinct ketamine induced increases in insula activation in response to social threat a. Randomized dose (X) (n=13 clinical participants).b.Altered states of anxiety (M) at the end of infusion (40 minutes).Example items are listed.A two-sided post-hoc paired t-test was used to compare the effect of placebo vs. 0.5mg/kg of ketamine on anxiety (T12 = 5.30, p < 0.001, Cohen's d = 1.53, 95% CI = [7.85,18.82]).We implemented an FDR correction to control for the testing of multiple scale subcomponents.c. Anterior insula engaged by threat faces (Y).The facial expression was extracted from Figure 1 of Williams et al., 2015 25 .The image was modified with permission from the developers of a database of 3D facial stimuli described in Gur et al., 2002 26 .d. Ketamine-induced effects (X) on reducing right anterior insula activity to threat faces (Y).An Averaged Causal Mediation Effect (ACME) mediation model was used to assess whether ketamine-induced anxiety mediates the effect of dose on acute changes in neural activity during emotional processing.No adjustments for multiple comparisons were made.e.Right anterior insula activity in response to threat faces as a function of fear/anxiety.A mediation model was used to analyze the data.Each bar graph displays the group mean and standard error (SE).+: p < 0.1, *: p <0.05, **: p < 0.01, ***: p<0.001.Source data are provided as a Source Data file.Abbreviations: fMRI = functional magnetic resonance imaging.

Table 1 .
. Comparison of post-hoc paired t-test results between the placebo, 0.05 mg/kg and 0.5 mg/kg conditions Abbreviations: ASCs = Altered States of Consciousness; SD = standard deviation; T = T score from paired t-tests; p = p value from paired t-tests; d = Cohen's d effect size from pairwise contrasts; *: p < 0.05; **: p < 0.01; ***: p < 0.001.Paired t-tests were two-sided.Source data are provided as a Source Data file.

Table 2 .
Summary of individuals' altered states of consciousness scores and their quotes at 20-30 minutes into the infusionEach measure is quantified as the change induced by ketamine at 0.5 mg/kg relative to placebo condition.Source data are provided as a Source Data file.
"I'm feeling weird, my body is tingling, hearing a hum, especially in my right ear.I've never felt this way before, neutral color all around.Chair legs, there is other stuff around but can only focus on the chair.

Table 3 .
Summary of results from mediation analysis

Table 5 .
Cannabis use in the past 7 days, alcohol use in the past 24 hours, and/or other illicit recreational drug use in the 48 hours prior to sessions.In addition to selfreport, these exclusion criteria will be assessed by a urine screen, saliva sample, or breathalyzer.Renal/hepatic impairment Hypertension (Hypertension, Stage 1 as defined by a systolic blood pressure >140 mmHg or diastolic blood pressure > 90 mmHg on two of three measurements at least 15 minutes apart at initial screening appointment; systolic blood pressure >155 mmHg or diastolic blood pressure >99 mmHg on two of three measurements at least 15 minutes apart during infusion visits).Demographics and baseline clinical depression and anxiety levels